Selenium is an essential micronutrient for human health. The major biological form of selenium, the amino acid selenocysteine (Sec), is found in 25 human selenoproteins and selenoenzymes, many of which play pivotal roles in basic biological processes. The mechanism of selenium and Sec incorporation into the protein is the central and yet unresolved problem of selenium biology. The goal of this proposal is to unravel key steps of this process at the molecular and structural level. Unlike the 20 standard amino acids, Sec is delivered to the ribosome by a specialized elongation factor, eEFSec in eukaryotes and SelB in prokaryotes. Interestingly, Sec is encoded by an in-frame UGA codon, otherwise a translational stop codon. Accurate decoding and differentiation of Sec and stop UGA codons in humans requires auxiliary protein (SBP2) and RNA factors (SECIS), which are absent in the canonical gene translation machinery. Selenium deficiency, mutations in selenoprotein genes, and low levels of selenoproteins lead to diseases affecting nervous, endocrine, muscular, immune, and reproductive systems. Failure to incorporate Sec diminishes selenoenzyme activity and compromises selenoprotein structure, allowing for disease development. The deletion of the eEFSec gene obliterates selenoprotein synthesis, and mutations in human SBP2 and SECIS cause pathologies. Although unrelated to the general translation elongation factors, the Sec-UGA decoding and Sec incorporation are still being described using the inadequate canonical model of protein synthesis. To address this deficiency, several critical aspects of the process will be addressed in this proposal. First, the mechanism by which eEFSec selects Sec-tRNASec among other aminoacyl-tRNAs will be determined using X-ray crystallography and in vitro binding and activity assays. Also, the proposed stringent selectivity of eEFSec towards Sec will be assessed by an in vitro translation system and by analyses of selenoproteins. Secondly, the structure of SBP2, its role in decoding, and the impact of pathogenic mutations on its structure and function will be determined. Given our preliminary findings on human eEFSec, the proposed work will shift the paradigm about processes regulating translation of selenoprotein genes and will significantly advance our understanding of the aspects of co-translational incorporation of selenium and Sec that are conserved across kingdoms.